Membrane‐Bound dd‐Carboxypeptidase and Transpeptidase Activities from Bacillus megaterium KM at pH 7

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Abstract
The membranes from Bacillus megaterium KM contained a DD‐carboxypeptidase with optimum activity under the following conditions: pH 7; ionic strength, 1.3 M; temperature, 40 °C and below 20 °C. It did not require any divalent cation, but was inactivated by Cu2+ and Hg2+. It was stimulated by 2‐mercaptoethanol and low concentrations of p‐chloromercuribenzoate. The membrane preparation also catalyzed a simple transpeptidation reaction using as carboxyl acceptors d‐alanine or glycine. The conditions for optimum activity, temperature‐inactivation, temperature‐dependence of the activity, carboxyl donor specificity, sensitivity to β‐lactam antibiotics, and insensitivity to potential peptide inhibitors of both enzyme activities, was identical. The dd‐carboxypeptidase showed inhibition by d‐alanine and Ac2‐l‐Lys‐d‐Ala. The inhibition by β‐lactam antibiotic was reversible for both enzymic activities and the time dependence for their recovery was identical. The dd‐carbozypeptidase was very sensitive to changes in the configuration and size of the side‐chains of the C‐terminal dipeptide of the substrate. Amino acid residues at the C‐terminus that precluded the peptide from being a dd‐carboxypeptidase substrate were not acceptors in the transpeptidation reaction. Dipeptides were not acceptors for the ‘model transpeptidase’. It is suggested that both activities are catalysed by the same enzyme molecule, whose physiological role is not the formation of peptide crosslinks during peptidoglycan biosynthesis.